Frequently Asked Questions
1. Why is simultaneous detection of multiple second messengers important?
Multiplex measurements in living cells tell us more about the biological response and may enable better assessments of potential adverse effects early in the drug discovery process. Cell-based calcium assays have been a mainstay of GPCR drug discovery and signal transduction research since the mid 1990s. However, a calcium readout alone is ambiguous. Many cellular processes produce calcium. Multiplex assays produce specific information about the signaling pathway and can indicate ligand bias.
2. Are genetically-encoded sensors robust enough for detection by fluorescence plate readers?
You can expect Z’ values of 0.6 and higher in transiently transduced cells.
3. What are some experimental parameters that I can change to optimize expression in my cells?
4. What advantages do Montana Molecular's cADDis cAMP assays have over GloSensor cAMP Assay from Promega?
How does cADDis cAMP assay compare with a cAMP assay that uses luciferase?
- cADDis is available with various promoter and enhancer systems for specialized cell types.
- cADDis is packaged in a variety of viral vector systems for optimized delivery to primary cultures and iPSC-derived cells.
- cADDis does not require the addition of luciferin to produce a signal.
- Well-validated protocols for detecting Gi signaling are available for the cADDis cAMP assay.
- Luciferase is known to interact with small molecules causing about 8-10% false positive “hits” in HTS. Chem Biol. 2012 Aug 24;19(8):1060-72.
- Red cADDis can be paired with green cADDis for ratiometric imaging of cAMP signaling.
- cADDis can be targeted sub cellularly, to compare cAMP levels in the organelle versus the cytosol.
- cADDis can be targeted to specific populations of cells via a cell-specific promoter systems including Cre-inducible expression
- cADDis can be multiplexed with other colored sensors, to measure two different signals, in the same cell, at the same time. Green cADDis paired with a red DAG sensors indicates when Gs/Gq bias is present. Red cADDis paired with Green ArcLight shows simultaneous cAMP and voltage signals.
5. How do I decide between an Upward DAG or cADDis assay and a Downward DAG or cADDis assay?
If you think of the signal as the absolute value of the change in fluorescence intensity, then the change in fluorescence intensity can be either increasing or decreasing regardless of whether the analyte is increasing or decreasing. We recommend the Upward sensors for imaging, because in imaging applications, the sensor is subject to more light, and the Upward signal is opposite of any potential bleaching effect. For plate reader applications, the signal is averaged over the well, so the Downward sensors are recommended because the signal from the sensor is opposite from any background fluorescence. However, both types of sensors work well in both imaging and plate reader applications. Let us know if you can’t decide as we can suggest some options for you.
6. Are Montana Molecular’s BacMam kits approved for use in my BSL-1 lab?
Yes. Our sensors are packaged in BacMam, Autographa californica, AcMNPV, which is a modified baculovirus. The virus in this kit is pseudotyped to infect mammalian cells. While the virus expresses in mammalian cells, the baculovirus genome is silent, and it cannot replicate to produce new virus in mammalian cells. While it should be handled carefully, in a sterile environment, it is classified as a Biosafety Level 1 (BSL-1) reagent.
7. What types of plate readers have been validated with your assays?
Our assays are compatible with automated fluorescent plate readers. Our customers have reported good results on:
- Hamamatsu FDSS
- Molecular Devices FLIPR
- Molecular Devices Flexstation
- Perkin Elmer Enspire
We have validated on:
- Biotek Synergy MX
- Biotek Cytation
- BMG CLARIOstar
- Epifluorescence microscopes
8. Do cADDis assays require forskolin ?
Our cADDis assays detect changes in cAMP in living cells by producing robust changes in fluorescence. As live cell assays, they are tuned to detect physiological changes in cAMP. At artificially high levels of cAMP, the sensor may saturate and become unresponsive. In other words, the cADDis assays are optimized for real time, kinetic measurement of biological levels of cAMP. Forskolin is a diterpene that dramatically amplifies the effect of Gs at adenyl cyclases. It also has direct effects on ion channels (Hoshi et al., 1988; Joost et al., 1988; Wagoner and Pallotta, 1988). Even a small amount of active Gs in a cell will produce large changes in cAMP if forskolin is present. This unique feature of forskolin has been used to assess “basal” levels of cAMP in unstimulated cells (Insel and Ostrom, 2003). In cAMP accumulation assays, forskolin is often used because higher levels of cAMP accumulation are easier to measure biochemically.
Using forskolin in our cADDis assays is not necessary or recommended as it disrupts the biological signals of the cell. If a GPCR with some basal level of Gs activity is present in the cell, forskolin will produce a very different response than one would see in a naive cell. Activation of the Gs-coupled receptor will also be greatly exaggerated by the presence of forskolin, and can easily produce cAMP levels that saturate the sensor. The apparent EC50 of agonists at the Gs-coupled receptor will be shifted by forskolin, making agonists appear more potent than they are. In the case of Gi-coupled receptors, forskolin’s effect on Gs stimulation is so profound that it is quite difficult to inhibit adenyl cyclase with Gi (Dessauer et al., 1998).
The bottom line is that the assay conditions for cADDis, a live cell assay, are very different from cAMP accumulation assays measured in a cell lysate. For example, poisoning the phosophodiesterase with IBMX will artificially raise cAMP levels to the point that cADDis would be saturated. Similarly, addition of forskolin can produce results that are difficult to interpret in the context of a live cell assay because the biological response of the cell is disrupted by its effect on the adenyl cyclase.
Dessauer, C.W., Tesmer, J.J., Sprang, S.R., Gilman, A.G., 1998. Identification of a Gialpha binding site on type V adenylyl cyclase. J. Biol. Chem. 273, 25831–25839.
Hoshi, T., Garber, S.S., Aldrich, R.W., 1988. Effect of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation. Science 240, 1652–1655. doi:10.1126/science.2454506
Insel, P.A., Ostrom, R.S., 2003. Forskolin as a Tool for Examining Adenylyl Cyclase Expression, Regulation, and G Protein Signaling. Cell Mol Neurobiol 23, 305–314. doi:10.1023/A:1023684503883
Joost, H.G., Habberfield, A.D., Simpson, I.A., Laurenza, A., Seamon, K.B., 1988. Activation of adenylate cyclase and inhibition of glucose transport in rat adipocytes by forskolin analogues: structural determinants for distinct sites of action. Molecular Pharmacology 33, 449–453.
Wagoner, P.K., Pallotta, B.S., 1988. Modulation of acetylcholine receptor desensitization by forskolin is independent of cAMP. Science 240, 1655–1657. doi:10.1126/science.2454507
9. Will BacMam work in my cell line?
BacMam has been validated in the following cell lines:
Link to BacMam transducible cells table
10. How do I change the standard protocol for cADDis and other assays if I need to transfect a receptor?
Example: Transfecting HEK293 cells with a receptor plasmid
Day 1: Seeding cells
Seed HEK 293 cells for transfection in 75cm2 flask. Aim for cells to be ≥80% confluent before transfection.
OPTION 1: cells can be seeded directly in 96 well plate, if so, follow Suggestions for Assays in Adherent Cells, described in the standard protocol.
Day 2: Transfect receptor:
- Ensure that cells are ≥80% confluent in flask before transfection.
- Tranfection of cells in the flask using lipofectamine2000 in OPTi-MEM.
- After 4 hrs change media. OPTION 2: transduction on Day 2, at least 1 hour after transfection.
Day 3: Transduction and Plating of cells according to the standard protocol.
Assay 24-36 hours post transduction.
11. What authentication steps are taken for BacMam stocks?
Our QA/QC processes are designed to make sure your assays are reliable and results are reproducible.
BacMam stock is tested for sterility before packaging. A 100 uL sample of each BacMam stock is added to 5 mL of rich cell culture media without antibiotic and is incubated at 37°C, 5% CO2, for five days and checked for bacterial or fungal growth.
Viral genes (VG) per milliliter (mL) are measured by qPCR with primers specific to VSVG. Viral samples are prepared to release viral genomic DNA, then multiple dilutions of the preparation are run in qPCR against a standard curve to generate an average titer for each BacMam stock. Each tube of stock is labeled with VG/mL and a stock keeping unit (SKU) identifier.
To test efficacy, serial dilutions are added to cultured HEK 293 cells. After 24 hours, the number of fluorescent cells are counted to establish transducing units per mL of stock.
BacMam is stable when stored at 4°C, protected from light. However, we recommend retesting BacMam stock after storing for more than 12 months at 4°C, or after any freeze-thaw cycle.
Sterile technique should be maintained at all times when handling BacMam stocks.